Methods and compositions are provided for gene therapy for pulmonary edema by the transfer of Na,K-ATPase subunit genes to lung epithelial cells for the purpose of increasing levels of Na, K-ATPase in vivo.
Cardiogenic and non-cardiogenic pulmonary edema affect millions of people each year causing substantial morbidity and mortality (Large State Peer Review, 1997). The alveoli of these people flood with liquid from pulmonary capillaries which compromises oxygen transfer to the systemic circulation (Hall et al., 1986). This sequence of events results in hypoxemia, hypercapnia, and death if no corrective measures are taken.
Unfortunately, no specific or satisfactorily effective treatment for pulmonary edema is available. Current therapy is entirely supportive and includes diuretic therapy to reduce pulmonary capillary hydrostatic pressure. This therapy has been shown to reduce edema accumulation but does not influence pulmonary edema clearance (Sznajder et al., 1986). In many cases, this therapy leads to inappropriately low left ventricular end diastolic volumes, reduced cardiac output, hypotension, and decreased peripheral oxygen delivery. Therapies that would improve or reconstitute the lung""s ability to keep itself dry could reduce the morbidity associated with pulmonary edema. (Matthay, et al., 1990, Verghese, et al., 1999)
Edema accumulates in the alveolus of the lung as a result of increases in capillary permeability and/or hydrostatic pressure, as described by Starling""s equation. (Staub, 1980) Conversely, edema is cleared from the alveolus as a result of active transport of Na+ out from the alveolar air space. This Na+ transport is due to the action of Na,K-ATPases that are located on the basolateral surface of alveolar type 2 epithelial (AT2) cells. These ATPases generate a transepithelial osmotic gradient that causes fluid movement out of the alveolar airspace via trans- and para- cellular pathways.
Lung edema clearance resulting from active Na+ transport has been demonstrated in live animal models, in isolated rat lungs, and in humans (Matthay and Wiener-Kronish, 1990; Effors et al., 1989; and Goodman et al., 1983). Supporting the role of active Na+ transport in lung liquid clearance are experiments in isolated rat lungs which demonstrate that lung liquid clearance is completely stopped by hypothermia (via inhibition of active transport), and is decreased by both amiloride (a Na+ channel inhibitor) and ouabain (a Na,K-ATPase inhibitor).
Na,K-ATPases are expressed in all eukaryotic cells where they are essential for the maintenance of cell volume and intracellular pH. They are also important for vectorial ion movement in many transporting and secretory epithelial throughout the human body. In particular, these ATPases are expressed in the alveolar epithelium where they reside on the basolateral aspect of alveolar type 2 cells.
Na,K-ATPase molecules are well controlled heterodimers. Endogenous Na,K-ATPase expression on the cell surface is tightly regulated and depends on changes in either intracellular Na+ or cell volume. As the stimulus for increased Na,K-ATPase activity abates, it is phosphorylated via protein kinase A (PKA) and/or protein kinase C (PKC) leading to internalization into late endosomes, creating cytoplasmic stores of assembled, potentially functional Na,K-ATPases. If needed, these Na,K-ATPase containing vesicles can be rapidly recruited to the cell membrane to meet immediate needs, should Na+ concentration or cellular volume change. In addition, intracellular pools of unassembled Na,K-ATPase subunit proteins exist in subcellular organelles and are also available for rapid assembly and recruitment to the cell membrane. Results of work by others suggest that there should be no need for additional, exogenous Na,K-ATPases.
Na,K-ATPases utilize high energy phosphates to exchange intracellular Na+ for extracellular K+. In addition to effecting vectorial Na+ movement, Na,K-ATPases regulate cell volume and intracellular pH and are responsible for transmembrane potentials in depolarizable cells. Functional Na,K-ATPase is composed of two subunits, (xcex1 and xcex2. Both subunits are required for normal Na,K-ATPase function. Three isoforms of each subunit have been identified and cloned. The xcex1 subunit has ATPase activity and is responsible for Na+/K+ exchange. The xcex2 subunit controls heterodimer assembly and life span, and trafficking to the plasma membrane. Although all cells express these proteins, isoform expression (e.g. xcex11/xcex21, xcex11/xcex22,xcex12,/xcex21) is developmentally regulated and differs between tissues. The xcex11, xcex12, and xcex21 subunits are the principal subunits expressed in rat lung.
Lungs of rats exposed to subacute hyperoxia (85% of O2xc3x977 days) have increased lung edema clearance (Olivera et al., 1994). These findings were associated with increased AT2 cell and whole lung Na,K-ATPase expression. Rats exposed to acute hyperoxia (100% of O2xc3x9764 hours) have decreased Na,K-ATPase expression and decreased lung liquid clearance. Thus, Na,K-ATPase expression and function parallel lung liquid clearance following hyperoxic lung injury.
Recombinant genetic technology has not been applied to treat lung injury. A possible approach is to use replication deficient adenoviruses which are useful for gene transfer. Adenoviruses are tropic for the respiratory epithelium, infect non-replicating cells with high efficiency, and do not integrate into the host genome. The absence of a crucial gene (E1a) makes it impossible for adenoviruses to replicate outside of cells that express E1a. Hence, adenoviruses do not propagate following infection of eukaryotic cells that do not express E1a. These recombinant vectors can be constructed with powerful promoters that allow high level, transient expression of a gene of interest in a cell transduced by adenoviruses.
A problem with the use of adenoviruses to transfer genes for therapy is that early (1st and 2nd) generation adenoviruses have been reported to cause significant host responses that limit their use for human gene therapy. These inflammatory effects are due, in part, to the expression of adenoviral antigens on the cell surface of transduced cells. These antigens cause a cytotoxic T-cell response that leads to elimination of the transduced cells. Suitable vectors for the delivery and short-term expression of many genes in the lung may be high-capacity, helper-virus dependent adenoviruses that contain no genes that express adenoviral proteins. Consequently it is expected that much of the anti-adenovirus host response will be abrogated by the use of these vectors, as such they are excellent. These vectors are also capable of gene transfer to cells in vitro. As such, these vectors are useful for the production of recombinant protein.
A goal is to use adenoviruses to develop gene therapy for lung illnesses, including pulmonary edema. Currently available treatments for pulmonary edema are unable to affect the lung""s ability to remove excess fluid from the alveolar airspace without affecting other organs. Thus, no specific treatments for pulmonary edema are currently available. The development of treatments that specifically affect pulmonary edema require the delivery of complex, functional proteins to alveolar epithelial cells. Currently there exists no pharmaceutical delivery system, other than gene transfer, that is capable of highly efficient overexpression of transport proteins such Na,K-ATPase in the alveolar epithelium.
Replication deficient adenoviruses have been previously used for human gene transfer studies. Most of these are phase I studies that have focused on the treatment of heritable conditions and cancer, and have yielded limited results. Gene therapy has not been reported for the treatment of acute or life threatening conditions. The use of these vectors for acquired conditions such as pulmonary edema would represent a new use for these vectors.
The invention relates methods and compositions for reducing pulmonary edema in acquired diseases of the mammalian lung using recombinant genetic technology. Adenoviruses are preferred vectors for the treatment of acquired, acute/short-term illnesses of the lung. A method of the invention includes the following steps:
(a) obtaining a recombinant genetic vector including
(i) an adenovirus that has no nucleotide sequences encoding adenovirus proteins; and
(ii) nucleotide sequences encoding Na,K-ATPase subunit genes that encode at levels that are an overexpression compared to levels in lung cells not having the genetic vector, and
(b) transferring the genetic vector into epithelial cells of the lung in vivo under conditions that allow expression of the subunit genes.
The invention also relates a recombinant genetic vector including:
(a) an adenovirus that has no nucleotide sequences encoding adenovirus proteins; and
(b) nucleotide sequences encoding Na,K-ATPase subunits at levels that are an overexpression compared to levels in lung cells not having the genetic vector.
An aspect of the invention is a host cell into which a recombinant genetic vector has been transferred, said vector including:
(a) an adenovirus that has no nucleotide sequences encoding adenovirus proteins; and
(b) nucleotide sequences encoding Na,K-ATPase subunits at levels that are an overexpression in vivo compared to levels in lung cells not having the genetic vector.
Suitable host cells include epithelial cells, in particular lung epithelial cells. Expression of genes from the vector in the host cell could take place in vitro or in vivo but the latter is preferred for clinical use. The present invention provides methods and compositions for the transfer of Na,K-ATPase subunit genes to lung epithelial cells and expression of the genes for the purpose of increasing in vivo Na,K-ATPase activity and improving lung liquid clearance. These methods and compositions are designed to augment endogenous alveolar transport processes for the purposes of gene therapy for pulmonary edema. Experimental data indicates that augmentation of Na,K-ATPase activity in vitro and in vivo requires overexpression of only one subunit (Factor, et al., 1998 a and b). The rate limiting( subunit varies among cell types, organs, and species. Selection of one of the three possible subunits is preferred and will vary between species and cell type. The use of a single subunit is desirable because it simplifies adenovirus construction and propagation, allows optimization of adenovirus design and minimizes cellular metabolic responses required to synthesize more than one transgene. For example, adenoviral-mediated gene transfer and expression of a ,xcex21 or xcex12 subunit gene increases Na,K-ATPase activity in rat cells and increases lung liquid clearance in rat lungs, whereas transfer of an xcex11 subunit gene is required to affect changes in active Na+ transport in human lung cells or monkey lungs.